IN SILICO ANALYSIS of FUNCTIONAL Snps of ALOX12 GENE and IDENTIFICATION of PHARMACOLOGICALLY SIGNIFICANT FLAVONOIDS AS
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Tulasidharan Suja Saranya et al. Int. Res. J. Pharm. 2014, 5 (6) INTERNATIONAL RESEARCH JOURNAL OF PHARMACY www.irjponline.com ISSN 2230 – 8407 Research Article IN SILICO ANALYSIS OF FUNCTIONAL SNPs OF ALOX12 GENE AND IDENTIFICATION OF PHARMACOLOGICALLY SIGNIFICANT FLAVONOIDS AS LIPOXYGENASE INHIBITORS Tulasidharan Suja Saranya, K.S. Silvipriya, Manakadan Asha Asokan* Department of Pharmaceutical Chemistry, Amrita School of Pharmacy, Amrita Viswa Vidyapeetham University, AIMS Health Sciences Campus, Kochi, Kerala, India *Corresponding Author Email: [email protected] Article Received on: 20/04/14 Revised on: 08/05/14 Approved for publication: 22/06/14 DOI: 10.7897/2230-8407.0506103 ABSTRACT Cancer is a disease affecting any part of the body and in comparison with normal cells there is an elevated level of lipoxygenase enzyme in different cancer cells. Thus generation of lipoxygenase enzyme inhibitors have suggested being valuable. Individual variation was identified by the functional effects of Single Nucleotide Polymorphisms (SNPs). 696 SNPs were identified from the ALOX12 gene, out of which 73 were in the coding non-synonymous region, from which 8 were found to be damaging. In silico analysis was performed to determine naturally occurring flavonoids such as isoflavones having the basic 3- phenylchromen-4-one skeleton for the pharmacological activity, like Genistein, Diadzein, Irilone, Orobol and Pseudobaptigenin. O-methylated isoflavones such as Biochanin, Calycosin, Formononetin, Glycitein, Irigenin, 5-O-Methylgenistein, Pratensein, Prunetin, ψ-Tectorigenin, Retusin and Tectorigenine were also used for the study. Other natural products like Aesculetin, a coumarin derivative; flavones such as ajoene and baicalein were also used for the comparative study of these natural compounds along with acteoside and nordihydroguaiaretic acid (antioxidants) and active inhibitors like Diethylcarbamazine, Zileuton and Azelastine as standard for the computational analysis. The protein-ligand interaction was studied and the docking analysis was performed by Patch Dock server using the 3D3L human arachidonate 12-lipoxygenase protein. Out of the 19 herbal constituents selected, it was found that Diadzein, Biochanin, Glycitein, Irigenin, 5-O-Methylgenistein, ψ-Tectorigenin and Tectorigenin showed significant binding affinity in inhibiting lipooxygenase enzyme as compared with standard compounds of acteoside, nordihydroguaiaretic acid, Diethylcarbamazine, Zileuton and azelastine. Naturally occurring compounds on further advancement could act as potent lipoxygenase inhibitors. Keywords: Cancer, Lipoxygenase enzyme, SNP, In silico analysis, Docking, Drug likeness INTRODUCTION partition coefficient) and Total Polar Surface Area (TPSA) Computer aided Drug designing is currently playing a major was calculated by the sum of fragment based contributions role in the field of research and drug discovery. The and correction factors4. TPSA is observed to be an conventional method of development of molecules by indispensable descriptor influencing the drug absorption, synthesis is preceded by the use of drug designing techniques CaCO2 permeability and blood-brain barrier penetration. computationally. Various imperative steps are involved in Molecular Volume calculation is predicted based on cluster drug designing such as structure based and ligand based drug contributions and are fully optimized by the semi empirical designing. In silico approaches based on ligand drug AM1 technique. Rule of 5 is a set of molecular descriptors designing have been performed for the lead molecule used by Lipinski in formulating the Rule of 5. The rule states development. The necessary molecular characteristics of the that most "drug-like" compounds have log P < = 5, molecular ligand have been generated computationally. The various weight < = 500, number of hydrogen bond acceptors < = 10 interactions between the proteins and the ligand are analyzed and number of hydrogen bond donors < = 55. Molecules by docking simulation with the protein target 3D3L1,2. The which do not pursue more than one of these rules may have binding is measured in terms of the score based on the problems with bioavailability. Topological parameter used for protein-ligand interactions. The study of other parameters measuring molecular flexibility is the Number of Rotatable such as detection of cavities was also carried out. Single Bonds (nrotb)6. Rotatable bond is defined as any single non- nucleotide polymorphism plays an essential role in ring bond, bounded to non-terminal heavy (non-hydrogen) identification of individual variations and determination of atom. The rule elucidates the molecular properties like mutations. Drug likeness is a parameter that helps to absorption, distribution, metabolism and excretion (ADME). characterize compounds based on the varied molecular Lipoxygenase (LOXs EC1.13.11) are iron containing properties like hydrophobic character, electron distribution, enzymes which are classified as arachidonate 5-lipoxygenase hydrogen bond formation, size and adaptability of the and 12-lipoxygenase in humans7. Lipoxygenase enzyme may molecule in conjunction with the presence of various initiate the synthesis of a signaling molecule or be involved pharmacophores. The lead molecule affects the in inducing structural or metabolic changes in the cell8. bioavailability, protein binding property, absorption Human lipoxygenases are the enzymes participating in the mechanism, toxicity and stability of the compound3. Mol metabolism of the polyunsaturated fatty acids and catalyzing inspiration was accustomed to calculate parameters like their oxidation to a variety of eicosanoids acting as the relative molar mass, log P, number of hydrogen bond donors secondary signal transducers playing a major role in human or acceptors which are essential to eliminate non-drug like homeostasis9. Phosphorylation via Mitogen activated protein molecules. Sophisticated Bayesian statistics was employed in kinase-2 is critical in controlling and activation of this Mol inspiration for the calculation of the physicochemical enzyme10,11. LOX are implicated in the pathogenesis of properties of the lead molecules. Log P (octanol/water diseases like asthma12, ulcerative colitis13, psoriasis14, Page 502 Tulasidharan Suja Saranya et al. Int. Res. J. Pharm. 2014, 5 (6) atherosclerosis15 and cancer16. In comparison with normal Aesculetin, ajoene, baicalein, along with the standard drugs tissues, significantly higher concentration of LOX like acteoside, nordihydroguaiaretic acid, intermediates have been found in breast, colon, lung, skin and diethylcarbamazine, Zileuton and azelastine were generated prostate cancers, along with persons with both acute and using Marvin Sketch and saved in the PDB format28. The chronic leukaemias17. Flavonoids are naturally occurring various properties of the ligands were studied using Mol plant metabolite with numerous pharmacological activities. inspiration. The ligands and proteins were subjected to The focus of the research was to verify certain selected energy minimization and finally the docking analysis was flavonoids as lipoxygenase enzyme inhibitor18,19. Certain performed using the Patch Dock server29. Patch Dock is an isoflavones like Genistein, Diadzein, Irilone, Orobol and algorithm for molecular docking. The input for Patch Dock is Pseudobaptigenin; O-methylated isoflavones such as molecules of any type like proteins, DNA, peptides or drugs. Biochanin, Calycosin, Formononetin, Glycitein, Irigenin, 5- The output is a list of potential complexes sorted by shape O-Methylgenistein, Pratensein, Prunetin, ψ-Tectorigenin, complementarily criteria30. The corresponding gene ALOX12 Retusin and Tectorigenin were selected for the study. The of the protein 3D3L has been explored to identify the isoflavones have the basic 3-phenylchromen-4-one skeleton functional effect of Single Nucleotide Polymorphism (SNPs) for the pharmacological activity. Other natural products like retrieved from dbSNP database31 and the deleterious amino Aesculetin, a coumarin derivative; ajoene and baicalein, acid substitution prediction by SIFT32. 696 SNPs have been flavone; were also used for the analysis. Acteoside and recognized in the ALOX12 gene, out of which 73 were in the nordihydroguaiaretic acid denoted as antioxidants and active coding non-synonymous region, 47 in the mRNA UTR inhibitors like diethylcarbamazine, Zileuton and azelastine region, 36 in the coding synonymous region and remaining were used as standard for the comparative computational 540 SNPs in the intron region of the gene. analysis. RESULTS MATERIALS AND METHODS The number of amino acids of the protein was found to be The X-ray crystallographic structure of lipoxygenase domain 541 with the molecular weight as 61089.2 g. The theoretical of human arachidonate 12-lipoxygenase, 12S-type (PDB ID pI was found to be 6.35 with the total number of negatively 3D3L) protein was obtained from RCSB protein data bank20 charged residues (Asp + Glu) as 58 and the total number of at a resolution of 2.60 Å. Water molecules, ligands and other positively charged residues (Arg + Lys) as 52. The total hetero atoms were removed from the protein molecule. number of atoms was found to be 8547. The Extinction Addition of hydrogen atoms to the protein was performed coefficient was 87735 at Abs 0.1 % (= 1 g/l) 1.436, assuming using CHARMm force field. The protein molecules were all pairs of Cys residues form cystines and Ext. coefficient at subjected to stability